The R2R3 MYB Transcription Factor DUO1 Activates a MaleGermline-Specific Regulon Essential for Sperm CellDifferentiation in Arabidopsis C W

Michael Borg,a,1 Lynette Brownfield,a,b,1 Hoda Khatab,a Anna Sidorova,a Melanie Lingaya,a and David Twella,2

a Department of Biology, University of Leicester, Leicester LE1 7RH, United KingdombDepartment of Biology and Zurich-Basel Plant Science Centre, Swiss Federal Institute of Technology, CH-8092 Zurich,

Switzerland

The male germline in flowering plants arises through asymmetric division of a haploid microspore. The resulting germ cell

undergoes mitotic division and specialization to produce the two sperm cells required for double fertilization. The male

germline-specific R2R3 MYB transcription factor DUO1 POLLEN1 (DUO1) plays an essential role in sperm cell specification

by activating a germline-specific differentiation program. Here, we show that ectopic expression of DUO1 upregulates a

significant number (;63) of germline-specific or enriched genes, including those required for fertilization. We validated 14

previously unknown DUO1 target genes by demonstrating DUO1-dependent promoter activity in the male germline. DUO1 is

shown to directly regulate its target promoters through binding to canonical MYB sites, suggesting that the DUO1 target

genes validated thus far are likely to be direct targets. This work advances knowledge of the DUO1 regulon that

encompasses genes with a range of cellular functions, including transcription, protein fate, signaling, and transport. Thus,

the DUO1 regulon has a major role in shaping the germline transcriptome and functions to commit progenitor germ cells to

sperm cell differentiation.

INTRODUCTION

In flowering plants, the process of male gametogenesis takes

place within specialized male reproductive organs, the stamens,

where meiosis occurs to produce haploid unicellular micro-

spores. Each microspore undergoes a highly asymmetric di-

vision to form a large vegetative cell encapsulating a small

generative or germ cell. The germ cell then undergoes a second

round of mitosis to produce two sperm cells. Upon successful

pollination, the vegetative cell grows a pollen tube through the

female stylar tissue to deliver the sperm cells to the embryo sac.

While one sperm cell fertilizes the egg cell to give rise to the

embryo, the other fertilizes the central cell to form the endo-

sperm. The production of fully differentiated sperm cells is thus

critical for double fertilization and has major implications for crop

fertility and seed production.

Although there have been significant advances in understand-

ing regulatory gene cascades in the sporophytic tissues that

nurture male gametophyte development, relatively little is known

about the scale and complexity of gametophytic regulatory

networks (Wilson and Zhang, 2009). In the male gametophyte,

a pioneering study established a late pollen regulatory network

governed by five pollen-specific MIKC* MADS box proteins that

is important in the vegetative cell for pollen maturation (Verelst

et al., 2007a, 2007b). A corresponding example in the female

gametophyte is a regulatory network modulated by the R2R3-

type MYB transcription factor MYB98, which regulates a battery

of synergid cell-expressed genes that are required for pollen tube

guidance and formation of the filliform apparatus (Punwani et al.,

2007). In terms of the gametes, transcriptional regulation is likely

to be an important aspect in germline development as plant male

germ cells have a distinct and diverse transcriptome (Engel et al.,

2003; Okada et al., 2006; Borges et al., 2008). Despite this, there

are currently no well-characterized regulatory networks de-

scribed in either the male or female plant germline.

DUO POLLEN1 (DUO1) was the first male germline-specific

transcription factor to be identified in plants, and mutation of

DUO1 results in a single mutant germ cell that is unable to

undergo fertilization (Durbarry et al., 2005; Rotman et al., 2005).

We have subsequently shown that DUO1 influences sperm cell

specification by regulating three male germline genes and inte-

grating their expression with germ cell cycle progression through

the G2/M phase-specific accumulation of CYCB1;1 (Brownfield

et al., 2009a). The three genes known to be regulated by DUO1

are MGH3 (HTR10), which encodes a male germline-specific

histone H3.3 variant (Okada et al., 2005; Ingouff et al., 2007),

GEX2, which encodes a membrane-associated protein (Engel

et al., 2005), and GCS1/HAP2, encoding an ancestral mem-

brane-associated protein required for gamete fusion (Mori et al.,

2006; von Besser et al., 2006; Hirai et al., 2008; Liu et al., 2008;

1 These authors contributed equally to this work.2 Address correspondence to twe@le.ac.uk.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: David Twell(twe@le.ac.uk).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.110.081059

The Plant Cell, Vol. 23: 534–549, February 2011, www.plantcell.org ã 2011 American Society of Plant Biologists

Steele and Dana, 2009; Wong and Johnson, 2010). DUO3 is

another regulatory protein that, like DUO1, is required to coor-

dinate germ cell division with gamete specification (Brownfield

et al., 2009b). DUO3 shares regulatory targets in common with

DUO1 since it is required for the expression of GEX2 and GCS1/

HAP2, although the precise reason for failed division in duo3

germ cells remains unknown as CYCB1;1 accumulation is unaf-

fected (Brownfield et al., 2009b).

Detailed analysis of DUO1 and the discovery of novel target

genes present a timely opportunity to uncover the scale and

organization of a germline transcriptional network that shapes

aspects of the sperm cell transcriptome and influences the

differentiation of the male gametes. We recently reported that

ectopic expression of DUO1 in seedlings results in the detection

of known DUO1 target transcripts MGH3, GEX2, and GCS1

(Brownfield et al., 2009a). Here, we exploit this inducible system

to screen for novel DUO1 target genes. We go on to describe

the validation of 14 of these target genes by demonstrating

DUO1-dependent promoter activity in the male germline and

transactivation in transient luciferase assays. We analyze the ex-

pression profiles of several DUO1 target genes during pollen

development and identify two promoters with sperm cell–specific

activity. Furthermore, we describe a series of experiments

that provide evidence that transactivation of DUO1 target genes

involves binding by the DUO1 MYB domain to conserved

sequences in target gene promoters and show that at least

one of these downstream genes is a direct DUO1 target.

Collectively, these data provide valuable insight into the down-

stream genes involved in the DUO1 male germline regulatory

network and significantly expand on the current models of male

gamete differentiation.

RESULTS

Novel DUO1 Targets Identified by Ectopic Expression

of DUO1

We previously reported transgenic lines that ectopically express

DUO1 in an estradiol-inducible manner and showed that ectopic

induction of DUO1 in seedlings results in the expression of three

known targets of DUO1: MGH3, GEX2, and GCS1 (Brownfield

et al., 2009a). This ectopic system utilizes mDUO1, a version of

theDUO1 cDNA that is resistant tomiR159 (Palatnik et al., 2007).

We exploited this inducible system to explore the scale of the

DUO1 regulon by screening for novel DUO1 target genes. We

performed comparative microarray analysis with RNA isolated

from transgenic seedlings that were transferred to plates with or

without inducer for 6, 12, or 24 h. RNA was isolated from three

biological replicates for uninduced and induced seedlings at

each time point and used to interrogate Affymetrix Arabidopsis

thaliana ATH1 genome arrays.

To identify putative DUO1 target genes, we applied several

filters to the data from the 24-h induction. Our first filter was to

only select genes that had a 3-fold change between the mean

expression values of the uninduced and induced replicates and a

statistically significant difference (Student’s t test, P value < 0.05).

Candidate targets were also limited to those with a present call in

at least two of the three induced samples. These criteria yielded

a total of 124 putative target genes (see Supplemental Data

Set 1 online). Given that DUO1 is specifically expressed in the

male germline (Rotman et al., 2005), its target genes should also

be expressed in sperm cells. Hence, our second filter was to

consider only those genes with a present call in sperm cell

transcriptomic data (Borges et al., 2008). Applying this criterion

narrowed down the final list of candidate DUO1 targets to 63

genes, including the known DUO1 targetsMGH3 and GEX2 (see

Supplemental Data Set 2 online). Herein, we will refer to our final

list of candidate genes as DUO1-activated target (DAT) genes.

The putative DAT genes were divided into three groups based on

their responses in the time course experiments (Figure 1A; see

Supplemental Data Set 2 online). Group A consists of 24 genes

that show a 3-fold increase between uninduced and induced

samples at all time points (6, 12, and 24 h). GroupB consists of 19

genes that have a 3-fold increase at 12 and 24 h, while Group C

contains 20 genes that show a 3-fold increase only after 24 h of

induction.

To gain a broad perspective of the expression of the DAT

genes, we referred to publicly availablemicroarray data. The vast

majority of rapidly responding genes in Group A, as well as

several of the later responding genes in Group B and C, appear

not be expressed in sporophytic tissues as indicated by the

MAS5.0-assigned absent calls in the array data (see Supple-

mental Data Set 2 online). To further illustrate tissue specificity,

we assessed whether DUO1-induced genes have sperm cell–

enriched expression according to the transcriptomic analysis of

Borges et al. (2008). Of the 63 DAT genes, nearly 80% show

sperm cell–enriched expression (see Supplemental Data Set 2

online). Interestingly, the earlier responding genes in Group A

contain the most sperm cell–enriched members (Groups A, B,

and C consist of 87.5, 78.9, and 65.0% sperm cell–enriched

genes, respectively). The number of sperm cell–enriched genes

in our list of putative targets is significantly increased compared

with the number occurring by chance in random lists of 63

genes called present in sperm (two-tailed x2 test: x2 = 6.03,

P < 0.05). These findings are consistent with the male germline-

specific expression of DUO1 (Rotman et al., 2005) and show it

induces transcription of predominantly male germline-enriched

genes.

MYBBindingSitesAreHighlyOverrepresented inCandidate

DUO1 Target Gene Promoters

If the candidate DAT genes are direct targets of DUO1, then their

upstream regulatory regions are expected to contain common

DNAmotifs bound by DUO1. To explore this possibility, the motif

analysis suite Regulatory Sequence Analysis Tools (van Helden,

2003; Thomas-Chollier et al., 2008) was used to identify any

overrepresented motifs in the promoter regions of our candi-

date DAT genes. Three highly overrepresented motifs (motif 1,

mwwAACCGTCwa; motif 2, awAAACCGCta; and motif 3,

adwAACCGTywh) were identified that each have a typical MYB

core containing the nucleotides AAC and are presented as a

position-specific scoring matrix (PSSM) in Figure 1B. Each

PSSM has high information content (9.7, 8.2, and 8.6, respec-

tively), showing a high sequence conservation within the PSSM.

Regulation of Sperm Cell Specification 535

A map of the distribution of these MYB binding sites (MBSs)

among our DAT gene promoters reveals a strong bias, with the

majority lying within the proximal 250 bp upstream of the start

codon (Figure 1C for validated candidates, see below; see

Supplemental Figure 1 online for remaining candidates). These

overrepresented MBSs are likely to be important for DUO1-

dependent transcription of target genes and suggest that DUO1

may activate some DAT genes directly. Consistent with this, the

earliest responding genes possess on average more MBSs in

their promoters than later responding genes, with Groups A, B,

and C containing 1.2, 0.5, and 0.3 MBSs, respectively (see

Supplemental Data Set 3 online). Furthermore, most of the genes

in Group A (19 of 24) have at least one MBS within 2250 bp of

ATG compared with Groups B (8 of 19) and C (6 of 20). Thus, the

rapid response of Group A genes may relate to the increased

number of proximal MBSs and direct activation by DUO1.

Figure 1. MYB Binding Sites Are Overrepresented in DUO1 Target Gene Promoters.

(A) DUO1 candidate targets divided into three groups based on their response time to ectopic DUO1 expression. Group A genes (black) have a 3-fold

increase after 6 h of DUO1 induction, Group B (blue) after 12 h, and Group C (red) only after 24 h of induction.

(B) Bioinformatic analysis of the promoters of candidate DAT genes identified three overrepresented MYB binding motifs. The consensus sequence of

the motifs is represented as a PSSM logo.

(C) A feature map encompassing positional information of the overrepresented MYB binding motifs in the 17 validated DAT gene promoters. The

colored boxes indicate the position of each MYB binding site with each color corresponding to the PSSM logos in (B).

536 The Plant Cell

CandidateDATGenesExhibitDUO1-DependentExpression

in the Male Germline

To validate candidate DAT genes and explore the specificity of

their expression, we generated stable transgenic H2B-green

fluorescent protein (GFP) marker lines driven by a selection of

DAT gene promoters and monitored their expression in the duo1

background (Figure 2). A total of 19 candidate target gene

promoters were tested, nine genes from Group A, five from

Group B, four from Group C, and an additional gene (At5g02390)

that failed the second selection filter (see Supplemental Data Set

2 online). The stringency of our selection criteria is likely to

exclude native targets that have a low and/or variable response

to DUO1. For example, GCS1 is a known regulatory target of

DUO1 (Brownfield et al., 2009a) but is not in our final list because

its response was variable between uninduced and induced

samples (see Supplemental Data Set 2 online). Like GCS1,

At5g02390 is called present in sperm cells (Borges et al., 2008)

and its induction was >3-fold, but this induction was unreliable

(see Supplemental Data Set 2 online). Thus, we included

At5g02390 in our analysis to confirm that we may have missed

target genes due to our strict criteria. For those target genes we

validated (see below) without an official gene symbol, we have

adapted the DAT nomenclature and named the genes according

to their predicted function (Table 1). For example, we termed the

protein encoded by At3g04620 DAN1 because it is a DUO1-

activated nucleic acid binding protein.

We first confirmed expression of each candidate promoter in

sperm cells by monitoring the nuclear-localized H2B-GFP re-

porter in mature pollen from several independent T1 lines. Of the

19 genes tested, three (At3g60780, At1g73510, and At5g08240)

showed no detectable expression in pollen, while one

(At5g45840) showed only vegetative cell expression (see Sup-

plemental Data Set 2 online), even although transcripts of these

four genes have been detected in Arabidopsis sperm cells

(Borges et al., 2008). This suggests that the promoter activity

of these genes is likely to be below the detection threshold of the

H2B-GFP reporter or that sequences downstream of these

promoters could be important for expression in sperm cells.

These four genes were thus excluded from further analysis. Of

the remaining 15 genes, 13 showed sperm cell–specific expres-

sion in mature pollen (Figure 2B; see Supplemental Data Set 2

online), while the remaining two (DAN1 and DAU1) showed

fluorescence in both the sperm cell and vegetative cell (Figure

2F; see Supplemental Data Set 2 online). Having established that

these 15 promoters were expressed in the male germline, we

analyzed their expression in duo1 germ cells.

Heterozygous duo1 plants produce 50%wild-type pollen with

two sperm cells and 50% duo1 pollen with a single germ cell

(Durbarry et al., 2005), enabling pollen genotype to be deter-

mined by germ cell number. Most lines displayed GFP fluores-

cence in >90% of wild-type sperm cells (Figures 2A and 2B).

However, several lines (DAF1, DAN1, DAU2, DAW1, OPT8, and

MAPKKK20) had a reduced percentage of wild-type sperm cells

with GFP fluorescence, which is likely due to a low GFP signal

resulting in a lack of detection in some sperm cells (Figures 2A

and 2E). For the 15 promoters analyzed, all but one (At5g60250)

showed a statistically significant reduction in the proportion of

germ cells with GFP fluorescence in duo1 compared with wild-

type pollen (Figures 2A and 2E). In duo1 germ cells little (Figures

2C and 2G) or no (Figures 2D and 2H) GFP fluorescence was

detected. For those promoters that also displayed vegetative cell

activity (DAN1 andDAU1), the GFP fluorescence was unaffected

in the vegetative cell of duo1 pollen (Figures 2E to 2H), with no

significant difference between the percentage of GFP-positive

vegetative nuclei in wild-type and duo1 pollen. These results

demonstrate that DUO1 is necessary for the activation of 14

promoters in the male germline and confirms them as DUO1

target genes (Table 1).

To complement the DUO1-dependent activity of DAT pro-

moters observed in the male germline, transient luciferase

assays were conducted in tobacco (Nicotiana tabacum) leaves

to determine whether DUO1 is sufficient for the transactivation of

target gene promoters in a heterologous system. Reporter

vectors were generated for 10 DAT genes (MGH3, GEX2,

GCS1, DAZ1, DAZ2, DAZ3, DAF1, ATOPT8, TIP5;1, and DAU1)

by fusion of the firefly luciferase open reading frame with each

target gene promoter (ProDAT:LUC). Infiltration of leaves with

Agrobacterium tumefaciens strains harboring the reporters alone

resulted in a low background level of relative luciferase activity

that is likely attributed to nonspecific basal transcription (Figure

2I). By contrast, coinfiltration of each ProDAT:LUC reporter strain

with a Pro35S:mDUO1 effector strain resulted in an increase in

activity that ranged from nearly 3-fold to >500-fold (Figure 2I).

This result further demonstrates that DUO1 is sufficient for the

transactivation of these target gene promoters in a heterologous

system, which implies that their activation is more likely to be

direct rather than through intermediate regulators.

Developmental Expression Profiles of DUO1 Target Genes

Previous analysis of DUO1 target genes has shown that the

DUO1 target genes MGH3, GEX2, and GCS1 are expressed

throughout male germline development soon after asymmetric

microspore division through to mature pollen (Brownfield et al.,

2009a). To determine if other DUO1 target genes show similar

profiles, we examined the developmental expression of a subset

of DAT genes, namely, TIP5;1, IMPa-8, DAA1, PCR11, and

DAN1. The activity of the TIP5;1promoter was typical of the other

three genes we have previously described (Brownfield et al.,

2009a), with GFP fluorescence absent from the microspore

(Figure 3A) and appearing specifically in the germ cell soon after

its inception and persisting in sperm cells of mature pollen

(Figures 3B to 3D).

In contrast with TIP5;1, IMPa-8 promoter activity was clearly

detectable at the microspore stage (Figure 3E). While the fluo-

rescence signal in the vegetative cell appears to decline following

microspore division (Figure 3F), it continues to accumulate in the

incipient germ cell and persists in sperm cells (Figures 3F to 3H).

This is consistent with DUO1-independent IMPa-8 expression in

the microspore followed by DUO1-dependent expression in the

germline, with a declining vegetative cell signal due to turnover

of inherited H2B-GFP protein. Unlike IMPa-8, no fluorescence

signal is detected at the microspore stage in DAN1 marker lines

(see Supplemental Figure 2A online), with promoter activity first

detected after microspore division in both the vegetative cell and

Regulation of Sperm Cell Specification 537

Figure 2. A Selection of Induced Target Genes Exhibit DUO1-Dependent Expression in the Male Germline.

538 The Plant Cell

the incipient germ cell (see Supplemental Figure 2Bonline).While

DUO1-independent DAN1 promoter activity appears to peak

midway through pollen development in the vegetative cell (see

Supplemental Figure 2C online), the DUO1-dependent activity in

the germline increases progressively and persists in mature

pollen (see Supplemental Figures 1C to 1F online).

Interestingly, the promoter activity of the two target genes

DAA1 and PCR11 appears to be sperm cell specific. A GFP

fluorescence signal in these marker lines was detected only after

germ cell division but not earlier in development (Figures 3I to 3L

for DAA1 and Figures 3M to 3P for PCR11). These genes both

belong toGroup B, which respond to DUO1 expression only after

12 h (see Supplemental Data Set 2 online), a delay that also

appears to be evident during male germline development. Col-

lectively, these observations highlight the diverse expression

patterns and complex regulation of male germline-expressed

DUO1 target genes during pollen development.

DAT Genes Belong to Several Functional Classes

To determine the range of functions potentially regulated by

DUO1, we undertook gene ontology (GO) analysis on the list of 65

candidate DAT genes. Although not part of our final list,GCS1 and

DAN1were also included since they are both validatedDAT genes

(Brownfield et al., 2009a; Figure 2E). Using the Classification

SuperViewer tool at the Bio-Array Resource (Provart and Zhu,

2003), the 65 genes were functionally classified according to the

three main GO categories (biological process, molecular function,

and cellular component) and the level of representation compared

with all Arabidopsis genes (see Supplemental Figure 2 online).

In terms of molecular function, transporter activity (GO:

0005215), hydrolase activity (GO:0016787), nucleic acid binding

(GO:0003676), and other binding (GO:0005488) are significantly

overrepresented in our list of DAT genes (see Supplemental Figure

3 online). Genes associated with the endomembrane system are

also highly overrepresented, with the endoplasmic reticulum

(GO:0005783) most notably being a significantly overrepresented

GO term (see Supplemental Figure 3 online). In parallel, we also

used the DAVID Gene Functional Classification tool (Huang et al.,

2008) to group this list of DAT genes into functionally related

classes based on significant co-occurrences of GO annotation

terms (see Supplemental Data Set 4 online). The enrichment

scores for the condensed groups were calculated by comparing

the 65 DAT genes with all genes called present in Arabidopsis

sperm cells (Borges et al., 2008). The low enrichment scores

indicate that no functional classes are highly enriched compared

with the sperm cell transcriptome, suggesting that DUO1 has a

wide role in sperm cell specification rather than regulating defined

pathways. Consistent with the idea that DUO1 regulates genes

important during male germline development, genes involved in

intracellular transport are enriched in both the sperm cell tran-

scriptome (Borges et al., 2008) and candidate DAT genes.

While not showing a significant overrepresentation in compar-

ison to the entire Arabidopsis genome, other functional classes

enriched in the sperm cell transcriptome are also present in our

list of DAT genes and include ubiquitin-degradation pathways,

ATPase activity, and G-protein signal transduction (see Supple-

mental Data Set 2 online). Although transcription factor activity

appears to be underrepresented in our DAT gene list compared

with theArabidopsis genome (see Supplemental Figure 2 online),

four putative transcription factors were induced, and three that

belong to the C2H2-type zinc finger family of proteins (DAZ1,

DAZ2, andDAZ3) have been validated as DUO1-regulated target

genes (Figure 2A).

MYB Binding Sites Are Critical for Transactivation of the

MGH3 Promoter

Having validated several DAT genes and identified overrepre-

sentedMBSmotifs in DAT promoter regions, we investigated the

Figure 2. (continued).

(A) Promoter activity of male germline-specific DAT genes. GFP expression was scored in single insert hemizygous lines in the duo1-1/+ background.

Wild-type (WT) pollen grains (black) were distinguished from duo1 pollen grains (white) by germ cell number. Each bar represents the mean of at least

three independent lines and error bars represent the SE. All 12 promoters show significantly reduced activity in duo1 germ cells compared with wild-type

sperm cells (two-tailed x2 test: P < 0.0001).

(B) to (D) Examples of male germline-specific DAT gene promoter activity in wild-type and duo1 pollen using CLSM. Each panel shows a representative

pollen grain under DAPI fluorescence (left) and GFP fluorescence (right). Sperm cell nuclei in wild-type pollen grains show a GFP signal (B), while duo1

germ cell nuclei show a residual level (C) or no detectable GFP signal (D). Arrowheads indicate a residual level of GFP signal in duo1 germ cells. Bars = 10mm.

(E) Promoter activity of DAT genes expressed in the vegetative and sperm cells. GFP expression was scored in single insert hemizygous lines in the

duo1-1/+ background for wild-type sperm cells (black), wild-type vegetative nuclei (dark gray), duo1 germ cells (white), and duo1 vegetative nuclei (light

gray). Each bar chart represents the mean of at least three independent lines, and error bars show the SE. The two promoters show significantly reduced

activity in duo1 germ cells compared with wild-type sperm cells (two-tailed x2 test: P < 0.0001), but there is no significant difference between activity in

the vegetative cells.

(F) to (H) Examples of non-male germline-specific DAT gene promoter activity in wild-type and duo1 pollen grains using CLSM. Each panel shows a

representative pollen grain under DAPI fluorescence (left) and GFP fluorescence (right). Both sperm cell and vegetative cell nuclei in wild-type pollen

grains show a GFP signal (F), while duo1 germ cell nuclei either show a residual level (G) or no detectable GFP signal (H), and the GFP signal of

vegetative cell nuclei is unaffected in duo1 pollen grains ([G] and [H]). Arrowheads indicate a residual level of GFP signal in duo1 germ cells. Bars = 10 mm.

(I) DUO1-dependent transactivation of validated DAT promoters in tobacco leaves. The relative luciferase activity (FLuc/RLuc) of each target promoter

(ProDAT:LUC) alone (�; light gray) and upon coinfiltration with Pro35S:mDUO1 (+; dark gray) is shown with the fold change indicated above the (+)

column. Each bar represents themean of at least four independent infiltrations, and error bars show the SE. A split y axis is presented in order to illustrate

lower level activity.

[See online article for color version of this figure.]

Regulation of Sperm Cell Specification 539

mechanism through which DUO1 regulates its targets. As a

model target promoter, we chose theMGH3 promoter since it is

strongly activated by DUO1 (Figure 2I; see Supplemental Data

Set 2 online) and contains five MBSs, four of which surround a

canonical TATA box within the 2250-bp region upstream of the

ATG start codon (Figure 4A). A 59 deletion series was analyzed

using transient luciferase assays to narrow down regions of the

MGH3 promoter required for DUO1-dependent transactivation

(Figure 4A). Deletions involving the removal of sequence up to

21082 bp, including theMBS furthest upstream of ATG, showed

a small reduction in relative luciferase activity (rLUC) compared

with the full-length promoter. A clear decrease in rLUC was

observed in deletion 3 (d3), where removal of a 1454-bp se-

quence reduced activity by ;30% (Figure 4A). The remaining

247 bp of promoter fragment has 69% the activity of the full-

lengthMGH3 promoter and contains four MBSs surrounding the

TATA box (Figures 4A and 4B). Further removal of the region

harboring the twoMBSs upstreamof the TATA box (deletion 4) all

but abolished rLUC (Figure 4A).

This result indicates that these proximalMBSs are likely to play

a major role in DUO1-dependent transactivation of the MGH3

promoter. This hypothesis was tested in context of d3 by

generating further deletions and promoter fragments in which

MBSs in this promoter fragment (termed A and B) were specif-

ically ablated by site-directed mutagenesis (Figure 4B). Muta-

genesis of MBS A (d3-mA) or B (d3-mB) reduced the rLUC by 67

and 73%compared with d3, respectively (Figure 4B). A fragment

mutating both these MBSs (d3-mAB) reduced the activity even

further, demonstrating that both MBSs act synergistically to

account for 93% of the rLUC of d3 (Figure 4B). This residual

activity is most likely due to the two remaining MBSs (C and D)

lying just upstream of the ATG start codon (Figure 4B). A deletion

fragment prior to MBS B (d3.5) reduced the rLUC by 81%

compared with d3, which is lower than that observed with the

d3-mA fragment (Figure 4B), indicating that the sequence con-

text surrounding the MBS is important for transactivation by

DUO1. Finally, mutagenesis of MBS B in context of d3.5 almost

abolished luciferase activity, showing just 3% activity of the

native d3.5 promoter (Figure 4B). These results demonstrate that

these MBSs are critical for DUO1-dependent transactivation of

the MGH3 promoter.

To determine whether the MBSs analyzed are necessary for

expression of MGH3 in the male germline, a two-way compar-

ison of promoter activity in mature pollen was performed be-

tween the activity of d3 of the MGH3 promoter (ProMGH3d3),

and the same deletion in which MYB sites A and B were both

mutagenized (ProMGH3d3_mAB). Both promoters were used

to drive expression of an H2B-GFP fusion and their activity

was monitored in wild-type plants. The GFP signal in the

ProMGH3d3:H2B-GFP lines (Figure 4C) was stronger than that

observed in the ProMGH3d3_mAB:H2B-GFP lines (Figure 4D).

To assess this difference quantitatively, a pooled pollen sample

Table 1. Summary of Validated DUO1 Target Genes

Groupa AGI Gene Name GO Protein

Promoter Activity

SC VC

A At1g19890 MGH3/HTR10b MALE GAMETE-SPECIFIC

HISTONE H3

Chromatin Histone H3 X

At2g17180 DAZ1 DUO1-ACTIVATED ZINC FINGER1 Transcription C2H2-type zinc finger protein X

At3g04620 DAN1 DUO1-ACTIVATED NUCLEIC ACID

BINDING PROTEIN1

DNA/RNA Nucleic acid binding protein X X

At3g47440 TIP5;1 TONOPLAST INTRINSIC PROTEIN 5;1 Transport Tonoplast intrinsic protein X

At3g62230 DAF1 DUO1-ACTIVATED F-BOX1 Protein fate F-box protein X

At4g35280 DAZ2 DUO1-ACTIVATED ZINC FINGER2 Transcription C2H2-type zinc finger protein X

At5g39650 DAU2 DUO1-ACTIVATED UNKNOWN2 Unknown DUF679 protein X

At5g49150 GEX2b GAMETE EXPRESSED2 Signaling Membrane protein X

At5g52000 IMPa-8 IMPORTIN a-8 Transport Importin a X

At5g53520 OPT8 OLIGOPEPTIDE TRANSPORTER8 Transport Oligopeptide transporter X

B At1g64110 DAA1 DUO1-ACTIVATED ATPASE1 Protein remodeling AAA+ type ATPase X

At1g68610 PCR11 PLANT CADMIUM RESISTANCE 11 Transport Zinc transporter X

C At3g50310 MAPKKK20 MITOGEN ACTIVATED PROTEIN

KINASE KINASE KINASE 20

Signaling Protein kinase X

At4g35560 DAW1 DUO1-ACTIVATED WD40 1 Protein fate WD40 protein X

At4g35700 DAZ3 DUO1-ACTIVATED ZINC FINGER3 Transcription C2H2-type zinc finger protein X

FSC At4g11720 GCS1/HAP2b GENERATIVE CELL

SPECIFIC1/HAPLESS 2

Signaling Membrane protein X

At5g02390 DAU1 DUO1-ACTIVATED UNKNOWN1 Unknown DUF3741 protein X X

AGI, Arabidopsis Genome Initiative.aGroups defined according to response time ($3-fold induction) in seedlings ectopically expressing DUO1 (A = 6 h, B = 12 h, and C = 24 h). FSC

indicates genes that failed the selection criteria.bGenes previously validated by Brownfield et al. (2009a).†Promoter activity determined in ProDAT:H2B-GFP marker lines by GFP signal in sperm cells (SC) and vegetative cell (VC) of mature pollen.

540 The Plant Cell

from six single locus lines was analyzed by microscopy and the

GFP fluorescence of sperm cell nuclei quantified. As shown in

Figure 4E, mutagenesis of both MBSs in ProMGH3d3_mAB

significantly reduced the GFP signal by almost 5-fold (Mann-

Whitney U test; U = 2768, P < 0.0001). These data demonstrate

that the MBSs present in the MGH3 promoter are necessary for

high levels of MGH3 expression in the male germline.

DUO1 Is Able to Bind to a MYB Binding Site in theMGH3

Promoter through Its R2R3 MYB Domain

To complement our findings that demonstrate the importance of

MBSs for DUO1-dependent transactivation, an electromobility

shift assay (EMSA) was performed to determine whether the

DUO1 MYB domain is able to bind to these MBSs in vitro. EMSA

assays were performed with recombinant DUO1 MYB domain

and an oligonucleotide containingMBS-A of theMGH3 promoter

(MBS; Figure 5A). DUO1 is able to bind this oligonucleotide in

vitro and this binding is competed with excess unlabeled MBS

competitor (Figure 5B). As a control, an additional oligonucleo-

tide was assayed in which the MBS was mutagenized (mMBS;

Figure 5A). DUO1 is unable to bind to mMBS, and an unlabeled

version of this oligonucleotide is unable to compete with binding

of DUO1 to the MBS oligonucleotide (Figure 5B). These bio-

chemical data demonstrate that DUO1 is able to bind specifically

to a MBS present in the promoter of the target gene MGH3.

We wanted to confirm that the interaction between DUO1 and

the MBS occurs through the DUO1 MYB domain. Thus, we

generated a variant cDNA in which the DNA binding capacity of

the MYB domain is compromised. Repeated Trp residues in the

MYB domain form a hydrophobic scaffold that maintains a helix-

turn-helix structure and DNA binding activity (Saikumar et al.,

1990), so a variant mDUO1 cDNA was generated by substituting

the second Trp of the R3 repeat at position 86 with a Gly residue

(mW86G; Figure 6A). To determine whether this mW86G variant

was able to function, its ability to transactivate the MGH3

promoter was compared with mDUO1 in transient luciferase

assays. To increase sensitivity of the assay, the effector cDNAs

had a disrupted miR159 binding site (mDUO1 and mW86G).

While coinfiltration with Pro35S:mDUO1 caused a 71.8-fold in-

crease in relative luciferase activity, coinfiltration with Pro35S:

mW86G resulted in only 2.7-fold induction (Figure 6B). This low

level of luciferase activity demonstrates that the integrity of the

DUO1 R2R3 MYB domain is important for wild-type levels of

MGH3 promoter transactivation.

To determine whether DNA binding capacity was essential for

DUO1 function in the male germline, the potential of the W86G

variant to complement the duo1 mutation was compared with

that of native DUO1. Expression constructs were created in

which DUO1 and W86G were fused to monomeric red fluores-

cent protein (mRFP) and driven by the DUO1 promoter to

generate ProDUO1:DUO1-mRFP1 and ProDUO1:W86G-mRFP

and stably introduced into duo1-1/+ plants homozygous for a

ProMGH3:H2B-GFP marker. As predicted in single locus lines,

the percentage of tricellular pollen expressing GFP in indepen-

dent T1 plants hemizygous for the ProDUO1:DUO1-mRFP trans-

gene (n = 5) was increased to;75% (Figure 6I), showing rescue

of the duo1 phenotype by restored mitotic division and GFP

Figure 3. Promoter Activity of DUO1 Target Genes during Male Game-

togenesis

Expression of ProTIP5;1:H2B-GFP ([A] to [D]), ProIMPa-8:H2B-GFP ([E]

to [H]), ProDAA1:H2B-GFP ([I] to [L]), and ProPCR11:H2B-GFP ([M] to

[P]) during wild-type pollen development as observed with fluorescence

microscopy. Representative pollen grains show GFP fluorescence (top,

green) and DAPI fluorescence (bottom, blue) at microspore (MSP), early

bicellular (EBC), late bicellular (LBC), and mature pollen (MPG) stages.

Bars = 10 mm.

Regulation of Sperm Cell Specification 541

expression in the half of the duo1 pollen that contains the

transgene (Figures 6C to 6E). Conversely, in plants hemizygous

for a single ProDUO1:W86G-mRFP transgene (n = 5), neither

mitotic rescue nor transactivation of the ProMGH3:H2B-GFP

marker line was observed (Figure 6I). This lack of complemen-

tation is supported by the detection of W6G-mRFP fluorescence

in GFP-negative duo1 germ cells in W86G lines (Figures 6F to

6H). These experiments provide robust evidence that DNA

binding capacity of the R2R3-MYB domain is essential for the

transactivation of DUO1 target genes in the male germline.

DISCUSSION

Here, we pioneered a detailed exploration of the plant germline

regulatory network that is modulated by the MYB transcription

factor DUO1. Building on our previous observations that DUO1 is

required for sperm cell specification, we have shown that DUO1

regulates the expression of a plethora of sperm cell expressed

genes by identifying 61 new candidate targets through the

ectopic expression of DUO1 in seedlings. Since we employed

strict criteria for the selection of candidate target genes, this

number is likely to be an underrepresentation. Indeed,GCS1 and

DAU1 both failed tomeet our selection criteria but were validated

as DUO1 target genes. Our strict selection criteria and the

demonstration of DUO1 dependence for themajority (13 of 14) of

candidate target gene promoters tested in themale germline give

us confidence that most candidates represent native DUO1

target genes. This has increased the number of validated DUO1

target genes from three to 17 and has identified nearly 50

additional genes that are likely to be DUO1 targets. This in-

creased knowledge of genes that form part of the DUO1 regulon

Figure 4. MYB Binding Sites Are Critical for DUO1-Dependent Transactivation of the MGH3 Promoter.

(A) Relative luciferase activity of 59 deletions in theMGH3promoter. Left shows the promoter sequences highlightingMYB sites (black boxes) and the canonical

TATA box (white box). Right shows the mean relative luciferase activity (FLuc/RLuc) for at least four independent infiltrations, and error bars show the SE.

(B) The significance of MYB sites for DUO1-dependent transactivation of the MGH3 promoter. Left shows the promoter fragments with targeted

mutations and/or deletions in context of d3 of the MGH3 promoter with MYB sites named A to D. Right shows the mean FLuc/RLuc for at least four

independent infiltrations, and error bars show the SE.

(C) Example of ProMGH3d3:H2B-GFP activity in mature pollen. Bar = 15 mm.

(D) Example of ProMGH3d3_mAB:H2B-GFP activity in mature pollen. A clear decrease in GFP signal was observed in several independent lines. Error

bars represent the SE. Bar = 15 mm.

(E) The decrease in GFP signal observed in ProMGH3d3_mAB:H2B-GFP lines. GFP fluorescence represents the mean total pixel intensity corrected

for background in (n) sperm cells analyzed. Asterisks indicate statistically significant differences determined using a two-tailed Mann-Whitney U test

(P < 0.0001, U = 2768).

542 The Plant Cell

reveals the functional diversity of pathways under DUO1 regu-

lation.

DUO1 Positively Regulates a Host of Functionally Diverse

Target Genes

During male germline development, a diverse array of genes is

expressed as shown by the unique transcriptional profile of

Arabidopsis sperm cells (Borges et al., 2008). DUO1 plays a

critical role in regulating sperm cell production, and as such its

downstream target genes are likely to be important for the

development of sperm cells as well as for fertilization. Our in-

vestigation of DUO1 target genes revealed that the majority are

preferentially expressed in the male germline and belong to

several functional classes (Table 1).

The apparent overrepresentation of transmembrane trans-

porter proteins in our list of candidate DAT genes (see Supple-

mental Figure 2 online) suggests that DUO1 is likely to play a

significant role in shaping this aspect of the sperm cell tran-

scriptome. Our analysis validated four DUO1-dependent trans-

port-associated genes (Table 1). PCR11 belongs to the PLANT

CADMIUM RESISTANCE family of genes, which encodes small

plasmamembrane proteins involved in the efflux of heavymetals

in Arabidopsis (Song et al., 2004, 2010). Another DUO1 target

that could have a role in metal transport is OPT8, one of nine

oligopeptide transporter (OPT) proteins in Arabidopsis. OPT

proteins have been implicated in the transport of diverse sub-

strates (Lubkowitz, 2006), with OPT3 functioning in metal ho-

meostasis and transport during Arabidopsis seed development

(Stacey et al., 2008).

TIP5;1 belongs to a subclass of the plant major intrinsic protein

family of aquaporins called tonoplast intrinsic proteins (TIPs;

Forrest and Bhave, 2007). TIP5;1 is involved in the transport of

water and urea (Vander Willigen et al., 2006; Soto et al., 2008) as

well as of boron in seedlings (Pang et al., 2010). TIP5;1 localizes

to mitochondria, and tip5;1 pollen tubes grow shorter in the

absence of exogenous nitrogen (Soto et al., 2010). Interestingly,

the TIP5;1 transcript is enriched in sperm cells (Borges et al.,

2008), and TIP5;1 promoter activity appears to be sperm cell

specific in mature pollen (Figure 3D). This suggests that sperm

cell function can influence pollen tube growth and that TIP5;1

could have an important role in nitrogen recycling within sperm

cells. Another DUO1 target involved in transport activity is IMPa-8,

which belongs to the importin a class of adaptor proteins

involved in nuclear protein import (Lange et al., 2007).

Protein metabolism is likely to be an important process during

germline differentiation, and as such, genes involved in ubiquitin-

mediated proteolysis are highly enriched in the sperm cell

transcriptome (Borges et al., 2008) and have an important role

in germ cell division (Kim et al., 2008). DUO1 appears to have a

role in regulating ubiquitin proteasome-dependent proteolysis

since the expression of DAF1, an F-box protein, is under DUO1

regulation. Another target, DAA1, belongs to a large superfamily

of ATPases that are involved in diverse processes, including

protein unfolding and degradation as well as in DNA recombi-

nation, replication, and repair (Snider and Houry, 2008).

The chromatin state of sperm cells, both its highly condensed

structure and its epigenetic marks, may be important for suc-

cessful karyogamy and subsequent development of the zygote

and endosperm (Ingouff et al., 2007; Berger et al., 2008). DUO1 is

known to regulate genes involved in chromatin structure since

the male gamete-specific histone H3 MGH3 (HTR10) is a DUO1

target gene (Brownfield et al., 2009a). Similarly, DAN1 has a

putative role in chromatin organization due its similarity with Alba

(Bell et al., 2002; Sandman andReeve, 2005), an archaeal protein

that affects the topology of chromosomal DNA in a temperature-

dependent manner (Xue et al., 2000).

DUO1 also regulates a number of putative transcription fac-

tors. Three of the validated target genes, DAZ1, DAZ2, and

DAZ3, all belong to the C2H2-type zinc finger family of transcrip-

tion factors (Englbrecht et al., 2004) and are among the most

highly expressed genes inArabidopsis sperm cells (Borges et al.,

2008). C2H2-type zinc finger transcription factors are known to

influence various developmental processes, including leaf/shoot

initiation (Takatsuji, 1998, 1999), floral organogenesis (Bowman

et al., 1992; Sakai et al., 1995), gametogenesis (Kobayashi et al.,

1998), and seed development (Gaiser et al., 1995; Meister et al.,

2002). The DUO1-dependent germline expression of three DAZ

genes (Figure 2) suggests that DUO1 is responsible for activating

subordinate regulons during male germline differentiation.

Cell-to-cell communication between pollen tubes and female

stylar tissues is critical during plant reproduction, and such

Figure 5. DUO1 Binds in Vitro to a MYB Binding Site in the MGH3

Promoter.

(A) Sequences of the oligos used in the EMSA experiments. MBS is the

region flanking MYB site A in the MGH3 promoter (bold and underlined),

and mMBS is a mutagenized version in which the MYB site has been

ablated (underlined).

(B) EMSA experiments using recombinant DUO1 MYB domain with MBS

or mMBS oligos. – and + indicate the absence or presence of the DUO1

MYB domain, respectively, and 3125, 3250, and 3250 indicate com-

peting unlabeled oligos. Addition of DUO1 MYB protein (lane 2 to 5)

causes a clear shift (white triangle) accompanied by a reduction in free

probes (black triangle), with free probes increasing in the presence of

more competing MBS oligos. In lanes 7 to 10, no shift was observed in

EMSA experiments using mMBS oligos. Results were confirmed in

independent EMSA experiments.

Regulation of Sperm Cell Specification 543

signaling processes appear to be at least partially under DUO1

regulation. DUO1 regulates the expression of GCS1/HAP2

(Brownfield et al., 2009a), an ancestral signaling molecule that

is required for efficient pollen tube guidance (von Besser et al.,

2006) and fertilization (Mori et al., 2006). Our demonstration that

MAPKKK20 is under DUO1 regulation (Figure 2A) suggests that

DUO1 also regulates mitogen-activated protein kinase (MAPK)

pathways in the germline. Interestingly, MAPKKK20 is phyloge-

netically related to FERTILIZATION-RELATED KINASE2 from

Chaco potato (Solanum chacoense), which is involved in seed

and fruit development (Gray-Mitsumune et al., 2006) as well as

pollen development and viability (O’Brien et al., 2007).

The DUO1 target gene DAW1 encodes a protein homologous

to the lethal giant larvae (Lgl) family of tumor suppressor pathway

genes, which play critical roles in cell polarization in a variety of

eukaryotic organisms (Baek, 1999; Bilder, 2001; Humbert et al.,

2003; Justice and Jan, 2003; Klezovitch et al., 2004). Recent

characterization of DAW1 in Arabidopsis extends Lgl conserva-

tion to the plant kingdom since DAW1 is able to partially substi-

tute for the function of yeast Lgl homologs (Forsmark, 2009).

Furthermore, daw1 mutations result in decreased lateral root

growth, consistent with a role for DAW1 in cell and tissue polarity

(Forsmark, 2009).

DUO1 Target Genes Are Differentially Expressed during

Male Germline Development

Not only does DUO1 appear to regulate proteins with a diverse

range of functions, but there also appears to be some variability

in the expression pattern of DUO1 target genes. We previously

reported that sperm cell specification begins early after inception

of the germline, with the expression of target genes mirroring

Figure 6. DNA Binding Integrity of the R3R3 MYB Domain Is Critical for DUO1 Function.

(A) Schematic diagram of the DUO1 protein with the R2 and R3 MYB repeats shown in blue. Below is the amino acid sequence of the R3 MYB repeat,

with the red and asterisked amino acids representing the hydrophobic scaffold. Below the native sequence (DUO1) is the variant sequence (W86G)

showing the mutation that compromises DNA binding capacity.

(B) Transactivation of the MGH3 promoter by mDUO1 and mW86G. Bars show the relative luciferase activity (Fluc/Rluc) of ProMGH3:LUC in tobacco

leaves with and without coinfiltration of Pro35S:mDUO1 and Pro35S:mW86G in at least four independent infiltrations, with error bars showing the SE.

(C) to (H)Mature pollen from transgenic lines expressing ProDUO1:DUO1-mRFP and ProDUO1:W86G-mRFP in duo1/+ plants homozygous for a ProMGH3:

H2B-GFP marker. Each panel from left to right shows the same pollen grains with DAPI, mRFP, and GFP fluorescence as illustrated below the images.

(C) to (E) Top left is an unrescued duo1 pollen grain with a bicellular phenotype (C) and no expression of the MGH3 marker (E). Bottom right is a fully

rescued pollen grain, with expression of ProDUO1:DUO1-mRFP (D) restoring tricellularity (C) and MGH3 marker expression (E).

(F) to (H) duo1 pollen grains expressing ProDUO1:W86G-mRFP (G) remain bicellular (F) and do not express the MGH3 marker (H).

(I) Ability of ProDUO1:DUO1-mRFP and ProDUO1:W86G-mRFP to rescue germ cell division (blue column) andMGH3 transactivation (green column) in

duo1 pollen. In duo1/+ plants (top columns),;50% of the pollen is wild type and has two GFP-positive sperm cells. Expression of DUO1-mRFP (middle

columns) rescues both germ cell division and activates MGH3 expression in the 50% of duo1 germ cells (25% total pollen) that contain the transgene.

Expression of W86G-mRFP (bottom columns) is unable to rescue germ cell division or transactivate the MGH3 marker line. Bars show the mean of at

least four hemizygous single locus lines, and error bars represent the SE.

[See online article for color version of this figure.]

544 The Plant Cell

DUO1 expression and beginning shortly after asymmetric divi-

sion of the microspore (Brownfield et al., 2009a). Our develop-

mental analysis of several DUO1 target genes has verified our

previous findings but also identified a novel class ofmale germline

genes that show sperm cell–specific expression (DAA1 in Figures

3I to 3L and PCR11 in Figures 3M to 3P). Interestingly, DAA1 and

PCR11 were induced above 3-fold only after 12 h of induction

(Group B; see Supplemental Data Set 2 online), mirroring the

delayed activation of these promoters in the germline. This

delayed response to DUO1 suggests that these promoters could

be subject to chromatin modifications and/or derepression only in

the presence of DUO1. The identification of sperm cell–specific

genes highlights the varied temporal patterns of gene expression

operating in themale germline and alludes to a spermcell–specific

differentiation program in which DUO1 plays a regulatory role.

The DAA1 and PCR11 promoters offer novel molecular tools for

the manipulation of gene expression specifically in Arabidopsis

sperm cells.

The use of previously assigned criteria to characterize the

sperm cell–expressed genes (Borges et al., 2008) indicates

that the majority of DUO1 target genes appear to be enriched

in sperm cells. However, in contrast with the male germline-

specific expression of DUO1 (Rotman et al., 2005), some DUO1

target genes show expression in sporophytic tissues and/or

other cell types of the male gametophyte (see Supplemental

Data Set 2 online). We have shown that two DUO1 target gene

promoters, DAN1 and DAU1, are active in the vegetative cell,

while the IMPa-8 promoter shows microspore activity that sub-

sequently becomes sperm cell specific. This indicates that

germline-independent mechanisms also operate to regulate

the expression of some DUO1 target genes. Consistent with

this, the expression of DAN1 and DAU1 in the vegetative cell is

unaffected in duo1 pollen grains (Figure 2E). It is likely that

parallel mechanisms involving other male germline regulators

also contribute to the activation of DAT genes as five DUO1

target genes (IMPa-8, DAZ1, DAZ2, DAN1, and DAU1) have a

residual level of expression in duo1 germ cells (Figures 2A and

2E). Indeed, germline expression of GEX2 and GCS1 has been

shown to be dependent on the regulator DUO3 (Brownfield et al.,

2009b). However, the low residual expression suggests that

DUO1 is the major factor required for the activation of these DAT

genes in the male germline.

DUO1 Binds to a MYB Consensus Sequence and Directly

ActivatesMGH3

An important aspect in defining a transcriptional network is to

determine whether the relationship between a transcription

factor and its target genes is direct or indirect. We identified

conserved MYB sites that occur frequently in the promoter

region of DAT genes (Figures 1B and 1C; see Supplemental Data

Set 3 online). The MYB sequences each contain a typical MYB

core AAC, which is bound by several other R2R3-type MYB tran-

scription factors, including C1 (Roth et al., 1991), P (Grotewold

et al., 1994), MYB.Ph3 (Solano et al., 1995), GAMYB (Gubler

et al., 1995), and MYB98 (Punwani et al., 2007). The majority of

MYB binding sites are present in the 2250-bp proximal region

upstreamofATGandareoftenadjacent tooneanother (Figure1C),

suggesting that this architecture may be an important feature of

DUO1 target promoters. We have shown that these MYB se-

quences are required for DUO1-dependent transactivation of the

MGH3 promoter (Figure 4). Furthermore, the DUO1MYB domain

is able to bind to one of these proximal MYB binding sites in vitro

(Figure 5), strongly suggesting that DUO1 directly activates

MGH3 through these motifs.

While theMGH3 gene appears to be a direct target of DUO1, it

is not known for certain whether this is the case for the other DAT

genes in this regulon. Most of the DAT genes, including the 16

validated DATs, contain at least oneMBSmotif in their promoter,

strongly suggesting that they too are direct DUO1 targets. Thus,

DUO1 appears to directly regulate numerous genes involved in

male germline development, and it is likely that the timing and

magnitude of activation depends upon factors such as the

architecture of the promoter region, the number and arrange-

ment of MBS motifs, and interactions with other regulatory

proteins. It is worth noting that other candidate genes upregu-

lated in seedlings in response to ectopic DUO1 expression do

not contain an MBS motif (see Supplemental Figure 3 online).

These genes may be indirectly regulated by DUO1 through

intermediate factors that are themselves under DUO1 regulation.

In conclusion, we uncovered an essential germline-specific

regulon that specifies major features of male germ cell fate in

Arabidopsis. The target genes that form the DUO1 regulon

highlight the range of functions required for sperm cell formation

as well as the unique characteristics integral to fertilization. In

particular, target genes like GCS1/HAP2, which is essential for

pollen tube guidance and fertilization, as well as TIP5;1, which

has a conditional role in pollen tube growth, demonstrate the

importance of the DUO1 regulon in sperm cell development and

function. Future work must endeavor to define the regulatory

networks operating in the male germline by uncovering the full

complement of genes regulated byDUO1 and other key germline

determinants like DUO3. The analysis of downstream regulatory

proteins, including the zinc finger proteins identified in this work,

coupled with comparative analysis in other species like rice

(Oryza sativa), will help to establish robust models of the regu-

latory networks that modulate male gamete differentiation in

plants.

METHODS

Plant Material and Transformation

Arabidopsis thaliana plants were grown at 218C with a 16-h-light and

8-h-dark cycle or with 24 h light (120 to 140 mmol/m2/s) with variable

humidity. Marker lines were generated in the +/duo1-1 Nossen-0 back-

ground or inwild-typeColumbia-0 for ProMGH3d3 andProMGH3d3_mAB

marker lines. Plants were transformed with Agrobacterium tumefaciens

(GV3101) using a standard floral dipping method (Clough and Bent, 1998).

Transformants were selected on soil with 30 mg/mL BASTA (glufosinate

ammonium; DHAI PROCIDA) fed by subirrigation or on Murashige and

Skoog agar containing 50 mg/mL kanamycin.

Microarray Analysis

Comparative microarray analysis was performed with RNA isolated from

transgenic 12-d-old seedlings harboring pMDC7-mDUO1 and grown in

Regulation of Sperm Cell Specification 545

the presence and absence of inducer for 6, 12, and 24 h as described by

Brownfield et al. (2009a). RNA was isolated from three biological repli-

cates using an RNeasy Mini Kit (Qiagen) and hybridized to Affymetrix

Arabidopsis ATH1 genome arrays at the NASCarrays facility. The raw

expression data generated from these 18 experiments are available

through the NASCarrays repository. The expression data were normal-

ized using the MAS5.0 algorithm and scaled with a 2% trimmed mean.

Data from triplicate experiments were used to calculate a mean expres-

sion value for each probe set, and the fold change between the uninduced

and induced values determined at each time point.

Vector Construction

Single and multisite Gateway recombination (Invitrogen) was used to

generate vectors as described by Brownfield et al. (2009a). The promoter

region of candidate genes was amplified from Columbia-0 genomic

DNA. The firefly and Renilla luciferase open reading frames were ampli-

fied from pRT2LUC (Man-Kim Cheung) and pK82 (Ralph Panstruga),

respectively. DUO1 variants were amplified from existing clones (Rotman

et al., 2005) or from amiR159-resistant DUO1 cDNA (Palatnik et al., 2007).

PCR reactions were performed with high-fidelity Phusion DNA polymer-

ase (Finnzymes) and primers with suitable attachment (attB) adapters

(see Supplemental Data Set 5 online; attB adapters in bold). Promoter

fragments were cloned into pDONRP4P1R and coding regions into

pDONR221 and verified by sequencing. The entry clones generated

and those described by Brownfield et al. (2009a) were recombined using

Multisite Gateway LR reactions with LR Clonase II Plus (Invitrogen) into

the destination vectors pB7m34GW or pK7m24GW,3 (Karimi et al., 2005)

to generate ProDAT:H2B-GFP marker constructs and ProDAT:LUC lu-

ciferase reporter constructs, respectively. Pro35S:mDUO1 and Pro35S:

mW86G effector constructs and the Pro35S:RenLUC control construct

were generated by single-site Gateway recombination into pB2GW7

(Karimi et al., 2002) using LR Clonase II.

Microscopy Analysis

Mature pollen was stained with 4’,6-diamidino-2-phenylindole (DAPI) as

described previously (Park et al., 1998). For developmental analysis,

pollen from buds at different stages of development was teased out of

staged anthers with a hypodermic needle andmounted directly into DAPI

solution or 0.3 M mannitol. Fluorescence and confocal laser scanning

microscopy (CLSM) were performed using the methods and equipment

described by Brownfield et al. (2009a).

Marker Line Analysis

For analysis of H2B-GFP marker lines, mature pollen shed from;24 T1

lines were stained with DAPI and analyzed by fluorescence microscopy.

Representative duo1/+ lines with a reasonable GFP signal and an

apparent single locus line (i.e., having ;50% GFP-positive wild type)

were chosen for further analysis. Detailed counts of GFP-positive and

GFP-negative sperm and germ cells in the wild type and duo1 portion of

the population, respectively, in at least three independent T1 lines for

eachmarker analyzed. Formarkers also expressed in the vegetative cells,

counts of GFP-positive and GFP-negative vegetative cell nuclei in wild-

type and duo1 pollen were also performed.

Complementation Analysis

For analysis of complementation lines, mature pollen from ;48 T1 lines

was examined by fluorescence microscopy. The T1 lines were initially

screened to identify representative duo1/+ lines with a single insertion for

the transgene (i.e., having ;50% RFP-positive pollen grains) and a

comparative mRFP signal. For all of the lines analyzed, the frequency of

duo1 pollen grains was determined by scoring the number of bicellular

and tricellular pollen grains by DAPI staining. The ability of the variants to

transactivate the marker line was assessed by counting the frequency of

pollen grains expressing GFP. The efficiency of rescue of the cell cycle

defect was determined by expressing the number of observed rescued

tricellular pollen grains as a percentage of the scored population. Sim-

ilarly, the efficiency of the variants to transactivate theMGH3marker line

was determined by expressing the number of GFP-positive pollen grains

as a percentage of pollen grains scored for GFP expression.

Quantification of GFP Fluorescence

For quantification of GFP fluorescence in ProMGH3d3:H2B-GFP and

ProMGH3d3_mAB:H2B-GFP marker lines, mature pollen from ;24 T1

lines were examined by fluorescence microscopy to identify six repre-

sentative single locus lines (i.e., having ;50% GFP-positive pollen

grains). A pooled pollen sample from the six representative lines was

analyzed using a Nikon ECLIPSE 80i fluorescence microscope using a

DS-QiMc cooled CCD camera (Nikon) and Plan Fluor 360/1.25 NA oil

immersion objective. The GFP fluorescence of sperm cell nuclei was

quantified in randomly selected pollen grains by image capture under

standardized conditions. The exposure time was determined empirically

to avoid image saturation and kept constant during image capture of all

mutants or trangenic lines analyzed. NIS-Elements BR v3.0 software

(Nikon) was used to process the images and determine the total pixel

intensity (TPI) of manually defined regions of interest encompassing

sperm cell nuclei. This region of interest was duplicated and used to

measure the TPI of the cytoplasmic background within the same pollen

grain. True fluorescence of sperm cell nuclei was determined by sub-

tracting the cytoplasmic background TPI from the nuclear TPI and amean

TPI calculated for each marker line.

Transient Transformation of Tobacco Leaf

Argrobacterium-mediated transient transformation of Nicotiana tabacum

leaf was performed as described by Sparkes et al. (2006) with some

modifications. A single colony from a desired Agrobacterium strain was

used to inoculate 5 mL of Luria-Bertani medium containing appropriate

antibiotics and cultured to saturation overnight at 288C with vigorous

shaking (220 rpm). Cells were harvested from a 1.5-mL aliquot of the

overnight culture by centrifugation at 1000g, and the pellet was resus-

pended in 1 mL of infiltration medium (280 mM D-glucose, 50 mMMES, 2

mMNa3PO4·12H2O, and 0.1mMacetosyringone). The cells werewashed

by resuspending in another 1 mL of infiltration medium to remove traces

of antibiotic, and the OD600 was measured using a Pye Unicam PU 8650

spectrophotometer (Philips).

Agrobacterium solutions were then combined according to the exper-

iment at an OD600 of 0.1 for reporter and effector vectors and an OD600 of

0.02 for the Renilla luciferase control vector. Four- to six-week-old

tobacco plants were grown in greenhouse conditions but placed under

a white fluorescent lamp for 1 h before infiltration to ensure fully open

stomata. Generally the third and fourth largest leaves from the apical

mersitemwere chosen for infiltration. Each infiltration was performed four

times, on both sides of the midrib region on two separate leaves. The

Agrobacterium solutions were taken up in 1-mL syringes and the under-

side of the leaf prepared by gently rubbing a 0.5-cm2 region to remove the

cuticle. The syringe tip was then placed against the rubbed regions and

the Agrobacterium solutions gently infiltrated while directly supporting

the front of the leaf with a finger. Infiltrated areasweremarked and labeled

with a permanent black marker pen. Gloves were sprayed with 70%

ethanol in between infiltrations to prevent cross-contamination. Plants

were placed in a growth chamber under normal growth conditions and left

for 2 d. Infiltrated regions were excised and used in dual luciferase

assays.

546 The Plant Cell

Transient Luciferase Assays

Agrobacterium-mediated transient transformation of tobacco SR1

leaves was performed as described by Sparkes et al. (2006) with

modifications. A standard size of Agrobacterium-infiltrated leaf was

excised using a cork borer and ground in 500 mL of 13 Passive Lysis

Buffer (Promega) until extracts appeared homogenous. Leaf extracts

were centrifuged at 48C at 14,000g to pellet cell debris. The firefly

luciferase assay buffer (25 mM glycylglycine, 15 mMKPO4, pH 8.0, 4 mM

EGTA, 2 mM ATP, 1 mM DTT, 15 mM MgSO4, 0.1 mM CoA, and 75 mM

luciferin with final pH adjusted to 8.0) and Renilla luciferase assay buffer

(1.1 M NaCl, 2.2 mM Na2EDTA, 0.22 M KPO4, pH 5.1, 0.44 mg/mL BSA,

and 1.43 mM coelenterazine with final pH adjusted to 5.0) were prepared

as described by Dyer et al. (2000). Two 25-mL aliquots were separately

assayed with 200 mL of each assay buffer using a Clinilumat LB9502

luminometer (Berthold). Relative luciferase activity (FLuc/RLuc) was

calculated for each infiltration by dividing firefly luminescence (FLuc)

with Renilla luminescence (RLuc).

EMSAs

The MYB domain sequence of DUO1 (amino acids 1 to 122) was inserted

via Gateway recombination into pDEST-544 (Addgene plasmid 11519) to

create a 6xHis-NusA-DUO11-122 translational fusion. Recombinant pro-

teins were purified using Ni-NTA spin columns (Qiagen) under native

conditions and dialyzed against storage buffer (20% glycerol, 0.5 mM

DTT, 6mMMgCl2, 50mMKCl, 0.1mg/mL BSA, 10mMTric-Cl, and 1mM

EDTA, pH 8.0). Double-stranded oligos (Sigma-Aldrich) were labeled with

digoxigenin using a Roche DIG Gel Shift Kit (v2) according to the

manufacturer’s instructions. The binding reactions were performed

according to Punwani et al. (2007) in binding buffer (10% glycerol, 0.5

mM DTT, 6 mM MgCl2, 50 mM KCl, 0.1 mg/mL BSA, 10 mM Tris-Cl, and

1mMEDTA, pH 8.0) for 15min at room temperature. Each 20-mL reaction

contained 32 fM of DIG-labeled oligos, an appropriate concentration of

unlabeled oligos, 300 ng of recombinant protein, 500 ng poly[d(I-C)], and

500 ng poly[d(A-T)]. Binding reactions were incubated on ice for 5 min

and resolved on native PAGE gels in 0.53 TBE buffer. The oligos were

transferred onto Genescreen Plus Charged Nylon Membrane (Perkin-

Elmer) by electroblotting in 0.53 TBE and cross-linked with a UV

Stratalinker (Stratagene). The membrane was developed according to

Roche DIG Gel Shift Kit instructions and imaged using an ICCD225

intensified CCD camera (Photek).

Bioinformatic Analyses

Overrepresentedmotifs were identified in candidate DAT gene promoters

using the online suite Regulatory Sequence Analysis Tools using default

settings (van Helden, 2003; Thomas-Chollier et al., 2008). The 2700-bp

intergenic sequence upstream of the start codon of each DAT gene was

compared with a background model consisting of the intergenic up-

stream sequence of all genes in Arabidopsis. PSSM logos were drawn

usingWebLogo (Crooks et al., 2004). GO analysis was performedwith the

Classification SuperViewer tool (Provart and Zhu, 2003) and functionally

classified according to the three main GO categories: biological process,

molecular function, and cellular component. The DAVID gene functional

classification tool (Huang et al., 2008) was used to compare DAT genes

with all sperm cell–expressed genes (Borges et al., 2008) using the lowest

classification stringency setting.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the accession numbers

provided in Supplemental Data Sets 1 to 4 online.

Author Contributions

M.B., L.B., and D.T. conceived and designed the experiments. M.B., L.B.,

H.K., M.L., and A.S. performed the experiments. M.B., L.B., H.K., and

D.T. analyzed the data. M.B., L.B., and D.T. wrote the manuscript.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Feature Map of MYB Sites in Candidate DAT

Gene Promoters.

Supplemental Figure 2. Developmental Expression of the DAN1

Promoter during Male Gametogenesis.

Supplemental Figure 3. Gene Ontology Analysis of Candidate DAT

Genes.

Supplemental Data Set 1. Genes Upregulated in Seedlings in

Response to Ectopic mDUO1 Expression.

Supplemental Data Set 2. List of Candidate DAT Genes.

Supplemental Data Set 3. Occurrence of MYB Motifs in Candidate

DAT Gene Promoters.

Supplemental Data Set 4. Output from the DAVID Gene Functional

Classification Analysis.

Supplemental Data Set 5. Primers Used in This Study.

ACKNOWLEDGMENTS

We acknowledge David Honys (Czech Academy of Sciences, Prague,

Czech Republic) and Gael Le Trionnaire (University of Leicester, United

Kingdom) for the collection and normalization of publicly available

microarray data. We thank Man-Kim Cheung (University of the West

of England, Bristol, United Kingdom) and Ralph Panstruga (Max-Planck

Institute for Plant Breeding Research, Cologne, Germany) for the Firefly

and Renilla luciferase cDNA, respectively. We thank June Saddington at

the University of Leicester Botanical Gardens for support with plant

cultivation. We acknowledge the Biotechnology and Biological Sciences

Research Council (BBSRC) for funding this work as well as for

supporting M.B. through a BBSRC-funded PhD studentship.

Received November 8, 2010; revised January 7, 2011; accepted January

12, 2011; published February 1, 2011.

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Regulation of Sperm Cell Specification 549

DOI 10.1105/tpc.110.081059; originally published online February 1, 2011; 2011;23;534-549Plant Cell

Michael Borg, Lynette Brownfield, Hoda Khatab, Anna Sidorova, Melanie Lingaya and David TwellArabidopsisEssential for Sperm Cell Differentiation in

The R2R3 MYB Transcription Factor DUO1 Activates a Male Germline-Specific Regulon

 This information is current as of November 8, 2020

 

Supplemental Data /content/suppl/2011/01/13/tpc.110.081059.DC1.html

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